Inter-laboratory validation study of the ForenSeq™ DNA Signature Prep Kit

Area of Science:

• Forensic Science
• Molecular Biology
• Genetics

Background:

• Massively parallel sequencing (MPS) presents advantages over capillary electrophoresis (CE) for forensic DNA-STR analysis, offering higher discrimination power through sequence information and multiplexing capabilities.
• The EU-funded DNASeqEx project evaluates MPS technologies, including the MiSeq FGx and Ion S5™ platforms, for forensic applications.

Purpose of the Study:

• To conduct an inter-laboratory validation of the Forenseq™ DNA Signature Prep Kit (Primer Mix A) for Short Tandem Repeat (STR) analysis.
• To assess the kit's performance regarding concordance, reproducibility, sensitivity, and mixture analysis in forensic DNA profiling.

Main Methods:

• Validation of the Forenseq™ DNA Signature Prep Kit on MiSeq FGx and Ion S5™ platforms.
• Testing concordance with CE results and reference profiles.
• Evaluation of reproducibility across duplicates and laboratories.
• Sensitivity testing with DNA inputs ranging from 1 ng to 31.25 pg.
• Analysis of male-male and male-female mixtures at various ratios.

Main Results:

• Sequencing results demonstrated high concordance with CE, reference profiles, and reproducibility between laboratories and duplicates.
• First locus drop-outs were observed at DNA inputs of 63 pg (20-plex pool) and 125 pg (38-plex pool).
• Allele balance was optimal at DNA inputs of 250 pg or higher.
• The kit performed well with moderate mixtures (ratios from 1:1 to 1:20).

Conclusions:

• The Forenseq™ DNA Signature Prep Kit (Primer Mix A) is a reliable tool for forensic STR analysis using MPS.
• The kit shows excellent concordance and reproducibility, suitable for forensic casework.
• Awareness of sensitivity limitations at low DNA inputs (below 125 pg) is crucial for interpretation.